To our initial studies of the effects of macromolecular crowding on enzymatic DNA joining, we noted that T4 DNA ligase could be enormously stimulated by high concentrations of polymers. We have extend those studies to a variety of DNA substrates. The rates of blunt-end and cohesive-end ligation of DNA by T4 ligase are increased by orders of magnitude in the presence of high concentrations of a variety of nonspecific polymers such as polyethylene glycol, Ficoll, bovine plasma albumin, or glycogen. Blunt-end ligation of small self-complementary oligodeoxyribonucleotides is also stimulated. The use of polyethylene glycol 6000 in such systems is characterized in some detail. Conditions are suggested which either greatly increase the rate of formation of and size of linear ligation products or which allow smaller but significant improvements in the amounts of circular ligation products. A patent covering these results on polymer-stimulated enzymatic ligation of DNA was applied for on behalf of the government.